Kev sib cais thiab ua kom huv ntawm cov protein yog dav siv hauv kev tshawb fawb biochemistry thiab daim ntawv thov thiab yog ib qho tseem ceeb ntawm kev ua haujlwm.Ib qho eukaryotic cell muaj peev xwm muaj ntau txhiab cov proteins sib txawv, qee qhov muaj nplua nuj heev thiab qee qhov tsuas muaj ob peb daim ntawv.Yuav kom kawm ib yamprotein ntau, nws yog ib qho tsim nyog yuav tau ua ntej purify cov protein los ntawm lwm cov proteins thiab cov tsis muaj protein ntau molecules.

1. Salting-tawm txoj kev ntawmprotein ntau:

Cov ntsev nruab nrab muaj qhov cuam tshuam loj rau kev solubility ntawm cov protein.Feem ntau, nrog kev nce ntsev concentration nyob rau hauv qis ntsev concentration, solubility ntawm cov protein nce.Qhov no yog hu ua salting;Thaum cov ntsev concentration tseem nce ntxiv, Solubility ntawm cov protein txo qis mus rau qhov sib txawv thiab sib cais tawm ib qho tom qab.Qhov no tshwm sim yog hu ua salting tawm.

2. Isoelectric point stacking txoj kev:

Lub electrostatic repulsion ntawm cov khoom yog qhov tsawg tshaj plaws thaum cov protein yog zoo li qub, yog li cov solubility kuj yog qhov tsawg tshaj plaws.Cov ntsiab lus isoelectric ntawm ntau cov proteins sib txawv.Cov pH ntawm cov tshuaj txias tuaj yeem siv kom ncav cuag qhov isoelectric point ntawm cov protein Ua kom nws sib sau, tab sis txoj kev no tsis tshua siv ib leeg thiab tuaj yeem ua ke nrog txoj kev salting-tawm.

3. Dialysis thiab ultrafiltration:

Dialysis siv ib lub semi-permeable membrane cais cov proteins ntawm txawv molecular ntau thiab tsawg.Txoj kev ultrafiltration siv lub zog siab los yog lub zog centrifugal los ua dej thiab lwm cov kua dej me me dhau los ntawm cov khoom siv semi-permeable, thaum lubprotein ntautseem nyob ntawm daim nyias nyias.Koj tuaj yeem xaiv qhov sib txawv ntawm qhov pore los cuam tshuam cov proteins ntawm qhov sib txawv molecular hnyav.

4.Gel filtration method:

Kuj hu ua qhov loj cais chromatography lossis molecular sieve chromatography, qhov no yog ib txoj hauv kev zoo tshaj plaws rau kev sib cais cov protein sib xyaw raws li molecular loj.Cov khoom ntim khoom siv ntau tshaj plaws nyob rau hauv kem yog qabzib gel (Sephadex ged) thiab agarose gel (agarose gel).

5.Electrophoresis:

Nyob rau hauv tib lub pH mob, ntau cov proteins tuaj yeem sib cais vim lawv qhov hnyav molecular sib txawv thiab cov nqi sib txawv hauv qhov hluav taws xob.Nws yog ib qho tsim nyog yuav tsum tau them sai sai rau isoelectric teeb electrophoresis, uas siv lub ampholyte ua tus nqa khoom.Thaum lub sij hawm electrophoresis, cov ampholyte tsim cov pH gradient maj mam ntxiv los ntawm qhov zoo electrode mus rau qhov tsis zoo electrode.Thaum cov protein nrog ib qho nqi ua luam dej hauv nws, nws yuav ncav cuag ib leeg.Lub pH txoj hauj lwm ntawm qhov taw tes hluav taws xob tsis tu ncua, thiab txoj kev no tuaj yeem siv los txheeb xyuas thiab npaj ntau yam proteins.

6.Ion kev sib txuas lus chromatography:

ion kev sib txuas lus muaj xws li cationic kev sib txuas lus agents (xws li carboxymethyl cellulose; CM-cellulose) thiab anionic kev sib txuas lus agents (diethylaminoethyl cellulose).Thaum dhau los ntawm ion kev sib txuas lus chromatography kem, cov protein nrog tus nqi rov qab rau tus neeg saib xyuas kev sib txuas lus ion yog adsorbed ntawm tus neeg saib xyuas kev sib txuas lus ion, thiab tom qab ntawd adsorbedprotein ntauyog eluted los ntawm kev hloov pH los yog ionic zog.

7.Affinity chromatography:

Affinity chromatography yog ib txoj hauv kev zoo heev rau kev sib cais cov proteins.Nws feem ntau yuav tsum tsuas yog ib kauj ruam los cais qee cov protein kom huv los ntawm cov protein sib xyaw nrog cov purity siab.

Txoj kev no yog ua raws li qhov tshwj xeeb es tsis yog covalent khi ntawm qee cov proteins rau lwm cov molecule hu ua ligand (Ligand).

Lub hauv paus ntsiab lus:

proteins muaj nyob rau hauv ib tug sib tov sib tov nyob rau hauv cov ntaub so ntswg los yog hlwb, thiab txhua hom ntawm tes muaj ntau txhiab tus proteins sib txawv.Yog li ntawd, qhov sib txawv ntawm cov proteins yog ib feem tseem ceeb ntawm biochemistry, thiab nws tsis tau nyob ib leeg.Los yog cov txheej txheem npaj ua tiav tuaj yeem tshem tawm txhua yam ntawm cov protein los ntawm cov protein sib xyaw, yog li ob peb txoj hauv kev feem ntau siv ua ke.